This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. The objective of the proposed research is to identify small RNAs expressed in four different developmental stages of Arabidopsis male gametophyte. Sexual reproduction in plants relies on the proper function of male and female gametophytes. Pollen, the male gametophyte, is responsible for producing two sperm cells and delivering them to fertilize the egg and two central cells in the embryo sac. Therefore, understanding the molecular mechanisms in the male gametophyte development in higher plants is important for many aspects of human life including agriculture and conservation. RNA silencing plays important roles in maintaining genome stability, controlling developmental processes, and helping the host to defend against the invasion of exogenous DNAs. In the RNA silencing pathway, small RNAs and associated protein complexes silence gene expression by cleaving messenger RNA, suppressing translation or modifying chromatin structure. Although the function of RNA silencing has been revealed in many plant developmental processes, we have almost no knowledge on how this pathway regulates gene expression in the male gametophyte. To better understand the role of RNA silencing in male gametophyte development, we propose to identify small RNAs expressed in four different developmental stages of the male gametophyte using cloning strategy. This project will be the first attempt to study small RNAs involved in pollen development. It will generate valuable information to direct future functional study on small RNAs and their target genes.